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Pompe disease is a glycogen storage disease caused by acid α-glucosidase enzyme deficiency. Currently, the unique treatment is lifelong enzyme replacement therapy ERT with frequent intravenous administration of the recombinant analog alglucosidase-α (AGA), which ultimately generates a sustained humoral response resulting in treatment discontinuation. Our aim is to use the tolerogenic properties of antigen-encapsulated red blood cells (RBCs) to abolish the humoral response against AGA and to restore tolerance to replacement therapy. To demonstrate that our approach could prevent the AGA-induced immune response, mice were intravenously injected three times with AGA encapsulated into RBCs before being sensitized to AGA with several adjuvant molecules. Control animals received injections of free AGA instead of the encapsulated molecule. One-week after treatment with AGA-loaded RBCs, a strong decrease in specific humoral response was observed despite three stimulations with AGA and adjuvant molecules. Furthermore, this specific immunomodulation was maintained for at least two months without affecting the overall immune response. AGA-loaded RBCs represent a promising strategy to induce or restore tolerance in Pompe disease patients who develop hypersensitivity reactions following repeated AGA administrations.

Erymet is a new therapy resulting from the encapsulation of a methionine gamma‐lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)‐dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U‐87 MG‐luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event‐free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half‐life was increased from

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

Asparaginase encapsulated in erythrocytes (ERY-ASP) is a potentially effective drug in patients with pancreatic adenocarcinoma (PAC) with null/low asparagine synthetase (ASNS) expression. Our aims were to assess ASNS expression in PAC from a large cohort and its prognostic and/or predictive value and to conduct a phase I trial with ERY-ASP in patients with metastatic PAC.Asparagine synthetase expression was evaluated using immunohistochemistry in resected PAC (471 patients) and in pairs of primary tumor and metastases (55 patients). Twelve patients were included in the phase I trial and received a single administration of ERY-ASP (25-150 IU/kg).Null/low ASNS expression was found in 79.4% of the resected PAC with a high concordance between primary tumor and metastases. Asparagine synthetase expression was significantly correlated with sex and CXCR4 expression. In the phase I trial, ERY-ASP was well tolerated by patients with metastatic PAC. No patient had DLTs, and 6 patients had at least 1 ERY-ASP causally related adverse event out of the 12 adverse events reported.Given the high rate of PAC with null/low ASNS expression and the good tolerability profile of ERY-ASP, ERY-ASP should be evaluated in further clinical studies in metastatic PAC.

Enterovirus RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.

Introduction: In current vaccine therapy, efficient delivery of tumor antigens (TA) to antigen-presenting cells (APC) represents a major step toward the development of strong TA-specific immune response including cytotoxic T lymphocyte (CTL) induction. Our innovative approach is to use the property of erythrocytes to be naturally phagocytosed by APC when senescent. Indeed, TA can be encapsulated into erythrocytes that will be used as carrier to specifically deliver the TA to APC, which will ensure their degradation and presentation to T cells. Materials and Methods: The proof of concept was established with Ovalbumin (OVA), tyrosinase-related protein 2 (TRP2 (melanoma TA) and prostate specific antigen (PSA, prostate TA). These TA were encapsulated into erythrocytes by a hypotonic lysis process. To stimulate the erythrophagocytosis by APC, the membranes of erythrocytes were coated with antibodies. Then, erythrocytes containing TA were intravenously injected to mice concomitantly with Poly(I:C) adjuvant. The CTL response was analyzed by IFNγ ELISPOT and in vivo lysis of TA-expressing target cells injected to immunized mice. Humoral response was assessed by the determination of TA-specific immunoglobulin titer in mouse serum. Results: All TA were efficiently encapsulated in erythrocytes in a dose-dependent manner. A strong T-cell response was induced after 2 injections of TA encapsulated in erythrocytes: 2μg of OVA and 20μg of TRP2 induced the in vivo lysis of 97% and 96% of TA-target cells, respectively. By comparison, injections of free antigen induced less than 5% of lysis. Furthermore, a significant number of TA-specific IFNγ-secreting cells was generated (1585 and 286 cells / 106 cells for TRP2 and PSA, respectively) compared to the free TA (< 80 cells / 106 cells for both TA). In addition, we established a dose-dependent PSA-specific humoral response. Finally, a significant delay of tumor growth was obtained in mice more than twenty days after the subcutaneous implantation of EG7-OVA or B16F10 (melanoma) tumor cell lines, compared to injections with free antigen or control vehicle. Conclusion: The use of erythrocytes as TA carrier to specifically deliver antigen to APC and induce efficient immune response against tumor can be a very promising strategy in cancer immunotherapy. Citation Format: Magali Cremel, Nathalie Guerin, Quitterie Barthe, Vanessa Bourgeaux, Willy Berlier, Françoise Horand, Yann Godfrin. Erythrocytes used as tumor antigen delivery system to target antigen-presenting cells embody an innovative approach for in situ cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2356.

Based on the asparaginase paradigm, several arginine-catabolizing enzymes have been developed for the treatment of arginine-dependent cancers (Wheatley, 2004). The arginine deiminase (ADI) enzyme catalyzes the hydrolysis of arginine (Arg) (Sugimura, 1990). ADI purified from Mycoplasma has a short half-life (≈4h) in the circulation and was found to be highly immunogenic (Holstberg, 2002). In order to increase half-life and to limit immunogenicity, a pegylated form of the enzyme (ADI-PEG-20) was developed. Phase I/II clinical studies in hepatocarcinoma and in advanced melanoma concluded on its limited efficacy at the tested doses. Notably, reduction of the duration of Arg depletion was linked to the emergence of ADI-PEG-20 antibodies (Ascierto, 2006). Encapsulation of ADI into red blood cells (RBC) is a promising alternative to improve the half-life and reduce the immunogenicity of the protein. Argininosuccinate synthase (ASS1) is the key enzyme in arginine biosynthesis (Haines, 2011). ASS1 expression varies in tumors and ASS1 loss is associated with poor prognosis in different cancers (Qiu, 2015). All these data strengthen the importance of selecting ASS1-negative patients for Arg-depletion based enzymatic therapy. Using a scalable, standardized production process, we synthesized an ADI enzyme from an optimized M. arginini coding sequence. ADI was encapsulated into RBC by hypotonic dialysis (ERY-ADI) and PK-PD parameters were evaluated in CD1 mice, in comparison with free ADI. Sensitivity to ADI was assessed in vitro by measuring the cell viability of 3 cancer cell lines with different ASS1 expression levels. ASS1 expression was screened by immunohistochemistry (IHC) in a large panel of tissue microarrays (TMA) from 16 human cancer types. Administration of ERY-ADI (5.5IU/mL) reduced mouse plasmatic Arg level to 30% of control values and led to a maintained depletion for 5 days. The same dose of free ADI strongly depleted Arg ( All these results highlight that arginine depletion through ADI treatment is effective against ASS1-negative cancer cells. ERY-ADI represents an innovative product with an improved efficacy for sustained Arg depletion and suitable for the treatment of ASS1 deficient cancers. Citation Format: Fabien Gay, Karine Aguera, Karine Senechal, Julie Bes, Anne-Marie Chevrier, Fanny Gallix, Christine Guicher, Philip Lorenzi, Vanessa Bourgeaux, Willy Berlier, Françoise Horand, Yann Godfrin. Arginine deiminase loaded in erythrocytes: a promising formulation for L-arginine deprivation therapy in cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4812.

Genetic differences between blood and mucosal-derived HIV-1 strains have been widely reported. As amplification of HIV-1 strains from mucosal samples including semen or saliva by co-culture has low sensitivity, we developed the construction of chimeric viruses expressing wild-type seminal HIV-1 envelope protein. Chimeric viruses were produced by co-transfection of a V1–V3 deleted pNL 43 vector and PCR fragments spanning the deleted region, amplified from HIV-1 RNA positive seminal plasma samples. After an initial testing of co-receptor usage by a tropism recombinant test, replication capacity and amplification of these recombinant viruses were assessed using PBMC. Four chimeric replicative strains, all using CXCR4 as coreceptor, were produced. The interaction between cell-free viral particles and reporter cell lines was assessed by confocal microscopy. These replicative chimeras exhibiting HIV-1 env from seminal strains represent useful tools for the in vitro study of the heterosexual transmission of HIV-1 and testing of microbicide activity.

The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

L-asparaginase (L-ASPA) is included in current chemotherapy regimen for the treatment of acute lymphoblastic leukemia (ALL). The sensitivity of ALL to L-ASPA has been linked to the incapacity of leukemic cells to produce their own asparagine, since they are deficient in a specific enzyme, asparagine synthetase (ASNS). Thus, ASNS-negative leukemic cells rely on plasmatic asparagine and can be starved to death by L-ASPA treatment. Several studies evidenced the potential of asparagine depletion to treat lymphomas. Indeed, L-ASPA is routinely administered to treat canine lymphomas and its adjunction in chemotherapy regimens significantly improves the outcome of patients with NK/T cell lymphoma. Some studies suggest its benefit for the treatment of B and T-cell non-Hodgkin lymphomas (NHL). In this study, we assessed the in vitro sensitivity to L-ASPA of 11 lymphoma cell lines, including diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL) and other NHL cell lines. We analyzed ASNS expression in tissue microarrays and biopsies from 226 cases of lymphomas, including 158 DLBCL, 15 PTCL, as well as other NHL and Hodgkin lymphomas. Sensitivity to L-ASPA (expressed as an IC50) was assessed by measuring the cell viability in the presence of various concentrations of L-ASPA. ASNS expression was assessed with a validated immunohistochemistry method attributing a score based on labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. All lymphoma cell lines were proved to be sensitive to L-ASPA. Their in vitro sensitivity usually exceeded well-known sensitive cell lines (MOLT-4 and HL-60) representative of acute leukemia. ASNS expression was null/low in 171/226 (76%) total cases of lymphomas. 118/158 (75%) DLBCL cases expressed no/low ASNS, whereas ASNS expression was low/null in 2/15 (13%) PTCL cases. Globally, B-cell NHL displayed a lower expression of ASNS than T-cell NHL, Hodgkin lymphoma and non tumoral lymph nodes. The in vitro sensitivity to L-ASPA of all lymphoma cell lines and the low/null expression of ASNS in the majority of the lymphoma tissues tested suggest that L-ASPA may be effective for the treatment of various lymphomas. As suggested by ASNS expression, L-ASPA treatment may be particularly effective in DLBCL and others B-cell NHL, whereas its efficacy in T-cell NHL is probably more limited. However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis…) render its use particularly hazardous in older or frail patients. The development of a new formulation of L-ASPA with better safety profile must be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Anne-Marie Chevrier, Alexandra Traverse-Glehen, Yann Godfrin. Low asparagine synthetase expression and in vitro sensitivity highlights L-asparaginase potential for the treatment of aggressive lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2015-5369

Background Heterosexual human immunodeficiency virus (HIV) transmission implies the crossing of the vaginal mucosa by virions present in the semen, potentially using Langerhans cells as transporters. The recruitment of these cells in the mucosa is mediated by the chemokine macrophage inflammatory protein 3alpha (CCL20). The aim of this study was to evaluate the capacity of the semen to induce Langerhans cell recruitment via the production of CCL20 by vaginal epithelial cells. Methods and results Using a vaginal epithelium model based on the SiHa cell line and human seminal plasma, we demonstrated that semen enhanced the production of CCL20. This secretion was regulated by the nuclear factor-kappaB intracellular signalling pathway. Fractionation of the seminal plasma indicated that the secretion of CCL20 was stimulated by high molecular weight compounds present in semen. Migration assays demonstrated that secreted CCL20 was able to promote the recruitment of Langerhans cell precursors (LCps), which remain permissive to X4 and R5 HIV infection. Conclusions Our results demonstrate that epithelial cells respond to factors present in semen by secreting CCL20, leading to the enhancement of LCp recruitment. These data argue in favour of the implication of epithelial cells in the heterosexual transmission of HIV.

L-asparaginase (L-ASP) is a well-established and important agent having demonstrated significant clinical efficacy in the treatment of acute lymphoblastic leukemia (ALL) Many cancerous cells are deficient in asparagine synthetase (ASNS), requiring endogenous plasma asparagine (ASN) for proliferation. Without this essential amino acid, the malignant cells undergo apoptosis. Plasma levels of ASN have been considered a relevant surrogate biological parameter to assess L-ASP efficacy. The aim is to maintain ASN levels below a threshold varying from 0.1 to 3µM. However, the measurement of ASN in presence of L-ASP is problematic, considering that 1 International Unit (IU) of L-ASP is able to cleave 1µmol of ASN per minute at 37°C. As the normal value of plasma ASN is around 50µM in humans, it requires about 30 seconds for 100 IU/L of L-ASP in the blood stream to fully deplete L-ASN, also considering there is no de novo ASN production., Plasma ASN is provided by normal cells expressing ASNS and by food intake. Once blood is drawn, no endogenous source of ASN is present and in the presence of active L-ASP a rapid ASN ex vivodepletion is expected to occur in the sampling tube. L-ASP activity can be reduced by low temperature (iced water) and/or by lowering the pH through addition of sulfosalicylic acid (Pieters et al, Blood, 2008). It is recommended to cool down the sample for 15 minutes on iced water immediately after the patient’s blood is drawn, and to centrifuge at 1000g for 15 minutes at +4°C to recover the plasma. A solution of 10% sulfosalicylic acid is added to the plasma 1:4 (v/v) before centrifugation at 3000g for 5 minutes to extract the proteins, including L-ASP. Here we report our investigation on the impact of the duration between blood draw and immersion in “iced water” and the impact of the sulfosalicylic acid concentration when “free” E. coliL-ASP (L-ASP) or L-ASP encapsulated in erythrocytes (L-ASP/RBC) is present. L-ASP or L-ASP/RBC was added to human blood, in concentrations comparable to patients receiving L-ASP treatment (0-200 IU/L). The sampling process used in clinical practice was strictly applied to the sample, except that different times (5 sec to 30 min) at room temperature (RT) between the addition of L-ASP (considered as the moment of the blood draw) and the step of cooling were tested. Two quantities (100µL and 500µL) of sulfosalicylic acid were also tested. ASN was quantified in the samples after the completion of the whole process to assess whether variations in these parameters could impact the measurement. When 20 IU/L of L-ASP was present in the sample, ASN was depleted by 75% after a 5-sec delay at RT, and below the limit of quantification (BLLQ=2µM) after 10 min. When reducing sulfosalicylic acid to a suboptimal quantity (100µL), complete depletion (BLLQ) of ASN was obtained after 5 sec at RT with 20 IU/L of L-ASP. These results confirmed the necessity of the acidic deproteinization of the samples but also revealed a significant ex vivo activity of L-ASP leading to overestimation of ASN depletion in plasma. Considering L-ASP/RBC, 25% ASN depletion was observed at 200IU/L after 3 min. at RT, and ex vivocomplete depletion was reached with 2000IU/L and a 30-minute delay at RT. In conclusion, considering current methods, due to inability to control ex vivo L-ASP metabolism, it is practically impossible to have a measurement of plasmatic ASN which reflects the in vivo reality. Indeed, in the presence of low concentrations of free L-ASP, a very rapid depletion of ASN is observed ex vivo. In the time needed to cool down and centrifuge samples further “artificial” depletion occurs rapidly and the patient can be erroneously considered ASN depleted. However, the comparison of the phamacodynamics between “free” asparaginases may be still considered valuable because the error is probably equal in both sides. On the contrary, “artificial” depletion is much less when the L-ASP is encapsulated in RBCs (thanks to the delay of asparagine to pass through the RBC membrane), leading to a potential bias to compare L-ASP or L-ASP/RBC. Finally, L-ASP activity is believed to be a more relevant marker than the measurement of ASN depletion. This is consistent with the approach taken by regulatory agencies in recent years, favoring the duration of L-ASP activity over 100IU/L as endpoint over depletion endpoints. Disclosures Berlier: ERYTECH: Employment, Equity Ownership. Godfrin:ERYTECH Pharma: Employment, Equity Ownership.

L-asparaginase (L-ASPA) displays a strong clinical benefit in the treatment of acute lymphoblastic leukemia (ALL), where it is included in most of current chemotherapy regimen. L-ASPA depletes plasmatic asparagine (ASN), an amino acid essential for the proliferation of leukemic cells. Since these cells are deficient in asparagine synthetase (ASNS), they rely on external (plasmatic) source of ASN and can be starved to death by L-ASP treatment. Several studies evidenced the potential of ASN depletion to treat lymphomas. Indeed, many animal and human lymphoma cell lines have been shown to be sensitive to L-ASPA in vitro. In veterinary medicine, L-ASPA is routinely administered to treat effectively both feline and canine lymphomas (Wypig et al ., 2013). L-ASPA regained attention in the treatment of human lymphomas since its adjunction in current chemotherapy regimens significantly improved the outcome of patients with NK/T cell lymphoma (Zou et al ., 2014). Some studies also evidenced its benefit in combined chemo or monotherapy for the treatment of B-cell and T-cell lymphomas (Sun et al ., 2006; Takahashi et al ., 2010). In this study, we assessed the in vitro sensitivity to L-ASPA of 6 lymphoma cell lines and we analyzed ASNS expression in biopsies from 166 cases of lymphomas (130 B-cell lymphomas and 17 T-cell lymphomas). Sensitivity to L-ASPA (expressed as an IC50) was assessed in vitro by measuring the cell viability in the presence of various concentrations of E.coli L-ASPA. ASNS expression in biopsies (TMA, USBiomax, Rockville, MD) was assessed with a validated immunohistochemistry (IHC) method attributing a score to each tumor based on ASNS labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. As shown in the following table, all cell lines were proved to be sensitive to L-ASPA. Their in vitro sensitivity exceeded cell lines MOLT-4 (ALL) and HL-60 (AML). | Cell line | Sensitivity to L-ASPA (IC50 in IU/mL) | | ----------------------------------------------- | --------------------------------------------- | | HuT-78 (Peripheral T-cell lymphoma,PTCL) | 0.11 ± 0.02 | | Toledo (Diffuse large B-cell lymphoma, DLBCL) | 0.19 ± 0.03 | | SU-DHL-8(Diffuse large B-cell lymphoma, DLBCL) | 0.10 ± 0.04 | | SU-DHL-10(Diffuse large B-cell lymphoma, DLBCL) | 0.10 ± 0.01 | | REC-1 (Mantle cell lymphoma, MCL) | 0.15 ± 0.03 | | KHYG-1 (NK/T-cell lymphoma) | 0.16 ± 0.06 | | MOLT-4 (acute lymphoid leukemia, ALL) | 0.19 ± 0.07 | | HL-60 (acute myeloid leukemia, AML) | 0.23 ± 0.02 | Table 1 As shown in the following table, ASNS expression was null/low in 85% in the entire population of patients with B-cell lymphomas. Considering DLBCL, 63% of patients displayed no ASNS expression at all. ASNS expression was also null/low in 88% of patients with T-cell lymphomas (n=17). | ASNS expression (IHC score) | Type of lymphoma (% of cases) | | ----------------------------------- | ------------------------------------ | ------------------ | | DLBCL (n=110) | Others BCL (n=20) | PTCL (n=3) | Others TCL (n=14) | MCL (n=3) | Hodgkin (n=16) | | Negative (0) | 62,7 | 70,0 | 0,0 | 57,1 | 33,3 | 43,8 | | Low positive (1) | 21,8 | 25,0 | 66,6 | 35,7 | 66,6 | 56,3 | | Positive (2) | 7,3 | 5,0 | 33,3 | 7,1 | 0,0 | 0,0 | | Highly positive (3) | 8,2 | 0,0 | 0,0 | 0,0 | 0,0 | 0,0 | Table 2 Globally, these results suggest that L-ASPA is potentially effective for the treatment of several lymphomas. Indeed, B-cell as well as T-cell lymphoma cell lines are sensitive to L-ASP in vitro and the majority of lymphoma tissues express no/low ASNS. Based on our results on ASNS expression in lymphoma biopsies, L-ASPA therapy may be beneficial for up to 85% of patients with DLBCL. Up to 90% of patients with other B-cell lymphomas or T-cell lymphomas may be sensitive to L-ASPA treatment as well. However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, and liver failure) render its use hazardous, particularly in older or frail patients. Therefore, the development of a new formulation of L-ASPA with safer profile has to be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Disclosures Berlier: ERYTECH: Employment, Equity Ownership. Aguera: ERYTECH: Employment. Chevrier: ERYTECH: Employment. Gallix: ERYTECH: Employment. Godfrin: ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background Cytokines present in the sperm could influence the heterosexual transmission of HIV by modulating viral titers and influencing the early immune response in the vaginal mucosa. Objectives To assess the relation between cytokine concentrations and HIV status in the seminal plasma. Study design Twenty-five HIV positive subjects were tested for cytokine content and HIV-1 load in seminal and blood plasma through a cross-sectional study. Results HIV positive subjects exhibited a significantly higher amount of seminal IL-1β as compared to a group of 33 HIV negative controls. In HIV positive subjects, amounts of IL-1β and HIV-1 RNA in semen were significantly correlated and a trend for correlation was found between seminal IL-1β and blood HIV-1 RNA. Amount of seminal IL-1β was significantly lower in patients under HAART, according to the decrease of their viral loads in blood and semen. Conclusions Considering that IL-1β is known to enhance viral replication and to promote immune response, its dosage in semen could represent an interesting marker for identifying patients at high risk for HIV heterosexual transmission.

Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3α/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1β through the nuclear factor-κB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

International audience; X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.

Enterovirus RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.

We have developed an in vitro reconstructed vaginal mucosa integrating Langerhans cells (LCs), obtained by differentiation of umbilical cord blood CD34 + hematopoietic progenitor cells, and, in this model, we have investigated the infection of LCs by human immunodeficiency virus type 1 (HIV-1). Proviral DNA of X4 (LAI and NL4-3) and R5 (Ba-L) HIV-1 strains were detected in LCs integrated in the reconstituted mucosa. Infection of LCs could be specifically inhibited by the chemokines stromal cell-derived factor 1 (SDF-1) and RANTES (regulated on activation, normally T cell-expressed and -secreted), confirming the presence of functional coreceptors on LCs generated in vitro. A complete inhibition of LCs, by use of azidothymidine, a reverse-transcriptase inhibitor, was also observed. Moreover, HIV-1-infected LCs of the reconstructed mucosa were able to transmit R5 or X4 HIV-1 strains to activated peripheral blood mononuclear cells. Such a model could be a useful tool to study the mechanisms involved in transmission of HIV in the female genital tract.

Due to asparagine synthetase (ASNS) deficiency, tumors cells auxotrophic for L-asparagine lack capability to synthesize this amino acid and depend to circulating L-asparagine for survival. Based on this rational, L-asparaginase (L-Aspa), an enzyme depleting L-asparagine, is an essential drug in the treatment of acute lymphoblastic leukemia (ALL), as these hematological cancer cells express low levels of ASNS. Recent evidence suggests that some solid tumors are also deficient in ASNS (Lorenzi, 2008; Dufour, 2012; Zhang, 2013), providing rationale for testing L-Aspa treatment in other contexts. In this study, we investigated the levels of ASNS expression in tumor tissues from patients with urological cancers (bladder, prostate and kidney) and we investigated the sensitivity to L-Aspa of bladder and prostate cell lines. Material & Method: ASNS expression was evaluated by a validated immunohistochemistry (IHC) method on tissue biopsie microarrays (USBiomax, Rockville, MD) including 384 cases of bladder, 94 cases of prostate and 75 cases of kidney tumors. A score was attributed to each tumor based on ASNS IHC labeling intensity from 0 (no labeling) to 3 (strong labeling). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. Sensitivity to L-Aspa (expressed as an IC50) was assessed in vitro by measuring the cell viability of bladder cancer cell lines (RT-4 and UM-UC-3) and prostate cancer cell line PC-3 in the presence of various concentrations of L-Aspa. ASNS expression in cell lines was evaluated by western-blot. Results: ASNS expression was low/null in 77.3% of bladder tumors, 86.2% of prostate tumors and 72% of kidney tumors. In bladder tumors, no correlation was found between the grade of the tumor and its expression of ASNS. Bladder and prostate cancer cell lines were all sensitive to L-Aspa (IC50 = 0.31, 0.12 and 0.22 IU/mL for RT-4, UM-UC-3 and PC-3 cell lines, respectively) at a level similar to the sensitivity observed with the L-Aspa-sensitive MOLT-4 ALL cell line (IC50=0.19 IU/mL). All cell lines displayed low basal expression of ASNS. Conclusion: The sensitivity of bladder and prostate cancer cell lines to L-Aspa and the low/no expression of ASNS in the majority of urological tumor biopsies suggest that an extensive population of patients with urological tumors may respond to asparagine depletion. However, L-Aspa is accompanied with well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, liver failure) and this drug should be used very carefully especially in older or frail patients. A new safer formulation is requested to make easier the clinical development of L-Aspa in urological cancer. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Fabien Gay, Gautam Borthakur, Yann Godfrin. Potential of asparagine depletion for the treatment of bladder cancer and other urological tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5436. doi:10.1158/1538-7445.AM2014-5436

Introduction The rational of use of L-asparaginase (L-Aspa) is based on asparagine synthetase (ASNS) deficiency in leukemic cells. Its efficacy is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis. It is a mainstay in the treatment of acute lymphoid leukemia (ALL), where it is established that resistance to treatment is in part related to detectable expression of ASNS (Aslanian et al., 2001; Su et al., 2008). In acute myeloid leukemia (AML), promising results have been obtained in clinical trials (Capizzi et al., 1988), with an improvement of complete remission rates from 18% to 54% in refractory patients 60 year old. More recently, it has been reported that leukemic cells from AML patients with M1, M4 and M5 FAB subtypes were more sensitive to L-Aspa (Okada et al., 2003).The aim of this study was to investigate the sensitivity to L-Aspa and the ASNS expression in an AML cell line (HL-60) and in primary leukemic cells from newly diagnosed AML patients. Materials and methods AML (HL-60) and ALL (MOLT-4) cell lines were grown according to ATCC recommendations. Primary leukemic cells from the bone marrow of 24 AML patients (median age: 69 years; range: 2-83) were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was determined by western-blot on isolated leukemic cells and expressed as a ratio ASNS/cyclophilin A. When a sufficient amount of leukemic cells was available, sensitivity to L-Aspa (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of L-Aspa (0.001 to 10 IU/mL) with the cells and by measuring the cell viability with a counting kit (CCK-8 viability assay) at day 4. Results Determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro (0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low but higher than in MOLT-4 which is a well known ASNS deficient cell line. IC50 determination was possible on 17/24 patients. Seven displayed a high sensitivity to L-Aspa (IC50 < 0.01 IU/mL) whereas 5 displayed a moderate sensitivity (IC50 < 0.5 IU/mL). Remaining 5 patients were resistant to L-Aspa. The cells from the healthy subject were resistant (IC50 > 10 IU/mL). To date, ASNS expression has been evaluated on 16 patients with ratio ranges from 0 to 2.27: six were negative, 4 low positive (< 0.2), and 6 positive (> 0.5, amongst them 4 were > 1). No correlation was observed between ASNS expression and FAB grade. However, patients with blasts sensitive to L-Aspa had mainly M1 or M5 AML. Conclusion AML cell line HL-60 and 71% of primary cells from AML patients were found sensitive to L-Aspa. The sensitivity of cells from M1 and M5 AML patients is consistent with the findings of Okada et al. (2003). Globally, these results suggest that L-Aspa is effective for killing AML cells. Based on the epidemiology of AML subtypes (Selter et al., 2011) and our results, L-Aspa therapy may be beneficial for 50-70% of patients with AML. However, L-Aspa has only been used scarcely in the treatment of AML, mainly because of the commonly observed adverse effects that impair its use. A new formulation of L-Aspa encapsulated in homologous red blood cells was reported with a better safety profile, allowing its use even in elderly patients (Hunault et al., ASH 2012). A clinical study is currently recruiting patients unfit for intensive chemotherapy in order to evaluate its efficacy in combination with low-dose cytarabine (NCT01810705) in AML. In this study, L-Aspa sensitivity and ASNS expression in primary tumor cells at diagnosis will be explored to investigate the relationship with clinical response. Disclosures: Berlier: ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Gallix:ERYTECH Pharma: Employment. Bertrand:ERYTECH Pharma: Principal Investigator Other. Thomas:ERYTECH Pharma: Principal Investigator Other; Orphan Europe: Consultancy. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.